Etienne E. Müller

Phenotypic and genetic characterization of the first two cases of extended-spectrum-cephalosporin-resistant Neisseria gonorrhoeae infection in South Africa and association with cefixime treatment failure

OBJECTIVES To describe the phenotypic and genetic characteristics of the first two cases of extended-spectrum cephalosporin (ESC)-resistant Neisseria gonorrhoeae in South Africa, one of which was associated with verified cefixime treatment failure. PATIENTS AND METHODS Two ESC-resistant N. gonorrhoeae isolates were cultured from the urethral discharge of two men who have sex with men (MSM). One man reported a persistent urethral discharge that had failed to respond to previous therapy with oral cefixime. Agar dilution MICs were determined for eight antibiotics. β-Lactam-associated resistance mutations were identified through PCR-based amplification and sequencing for several key genes: penA, mtrR and its promoter, porB1b (penB), ponA and pilQ. For molecular epidemiological characterization, full-length porB gene sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) were performed. RESULTS Both isolates were resistant to cefixime, ciprofloxacin, penicillin and tetracycline and intermediate/resistant to azithromycin, but susceptible to ceftriaxone, gentamicin and spectinomycin. Both isolates had the type XXXIV penA mosaic allele in addition to previously described resistance mutations in the mtrR promoter (A deletion), porB1b (penB) (G101K and A102N) and ponA1 (L421P). Both isolates had an identical NG-MAST sequence type (ST4822) and MLST sequence type (ST1901). CONCLUSIONS Both isolates were resistant to cefixime and possessed a number of identical mutations in key genes contributing to ESC resistance in N. gonorrhoeae. The two isolates contained the type XXXIV penA mosaic allele and belonged to a successful international MSM-linked multidrug-resistant gonococcal clone (MLST ST1901) associated with several cefixime treatment failures in Europe and North America.

Etiology and STI/HIV coinfections among patients with urethral and vaginal discharge syndromes in South Africa.

Background: This study was undertaken to establish the etiology of the male urethral discharge (MUDS) and vaginal discharge (VDS) syndromes, to determine the prevalence of other sexually transmitted infections (STI) and human immunodeficiency virus (HIV) coinfections, and to examine associations between STIs and HIV serostatus among STI patients in South Africa. Methods: A total of 507 MUDS and 300 VDS patients were recruited in Cape Town (CPT) and Johannesburg (JHB). A multiplex polymerase chain reaction assay detected Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and Mycoplasma genitalium infections. Bacterial vaginosis and candidiasis were detected by microscopy. Sera were screened for syphilis, HSV-2, and HIV antibodies. Results: Etiological diagnoses were made for 92% of MUDS patients and 85% of VDS patients. Gonorrhoea accounted for 85% (CPT) and 71% (JHB) of MUDS presentations. Chlamydia was the second most frequently detected MUDS pathogen (CPT, 13%; JHB, 24%). Among VDS patients, bacterial vaginosis was the most common cause (CPT, 46%; JHB, 36%) and trichomoniasis the most frequently detected STI pathogen (CPT, 19%; JHB, 34%). Few patients (4%) had serological evidence of syphilis. The HSV-2 and HIV seroprevalence were higher in Johannesburg compared to Cape Town and among women compared to men. HIV infection was statistically significantly associated with HSV-2 seropositivity at both sites and with the presence of N. gonorrhoeae and absence of C. trachomatis in Cape Town MUDS patients. Conclusions: Gonorrhoea and bacterial vaginosis were confirmed as the most frequent causes of MUDS and VDS. The high HIV seroprevalence in STI patients emphasizes the need to address HIV testing among this population.

Prevalence and Correlates of Mycoplasma genitalium Infection Among Female Sex Workers in Kampala, Uganda

BACKGROUND The importance of Mycoplasma genitalium in human immunodeficiency virus (HIV)-burdened sub-Saharan Africa is relatively unknown. We assessed the prevalence and explored determinants of this emerging sexually transmitted infection (STI) in high-risk women in Uganda. METHODS Endocervical swabs from 1025 female sex workers in Kampala were tested for Mycoplasma genitalium using a commercial Real-TM polymerase chain reaction assay. Factors associated with prevalent Mycoplasma genitalium, including sociodemographics, reproductive history, risk behavior, and HIV and other STIs, were examined using multivariable logistic regression. RESULTS The prevalence of Mycoplasma genitalium was 14% and higher in HIV-positive women than in HIV-negative women (adjusted odds ratio [OR], 1.64; 95% confidence interval [CI], 1.12-2.41). Mycoplasma genitalium infection was less prevalent in older women (adjusted OR, 0.61; 95% CI, .41-.90 for women ages 25-34 years vs <25 years; adjusted OR, 0.32; 95% CI, .15-.71 for women ≥ 35 years vs those <25 years) and in those who had been pregnant but never had a live birth (adjusted OR, 2.25; 95% CI, 1.04-4.88). Mycoplasma genitalium was associated with Neisseria gonorrhoeae (adjusted OR, 1.84; 95% CI, 1.13-2.98) and with Candida infection (adjusted OR, 0.41; 95% CI, .18-.91), and there was some evidence of association with Trichomonas vaginalis (adjusted OR, 1.56; 95% CI, 1.00-2.44). CONCLUSIONS The relatively high prevalence of Mycoplasma genitalium and its association with prevalent HIV urgently calls for further research to explore the potential role this emerging STI plays in the acquisition and transmission of HIV infection.

Association between Mycoplasma genitalium infection and HIV acquisition among female sex workers in Uganda: evidence from a nested case-control study.

Objectives Cross-sectional studies have shown a strong association between Mycoplasma genitalium and HIV infections. We previously reported that in a cohort of female sex workers in Uganda, M genitalium infection at baseline was associated with HIV seroconversion. Here we examine the temporal association between the M genitalium infection status shortly before HIV seroconversion and HIV acquisition. Methods A nested case-control study was conducted within a cohort of women at high risk for HIV in Kampala. Cases were those of women acquiring HIV within 2 years of enrolment. For each of the 42 cases, 3 controls were selected from women HIV negative at the visit when the corresponding case first tested HIV seropositive. The association between HIV acquisition and M genitalium infection immediately prior to HIV testing was analysed using conditional logistic regression. Results There was weak evidence of an association between M genitalium infection and HIV acquisition overall (crude OR=1.57; 95% CI 0.67 to 3.72, aOR=2.28: 95% CI 0.81 to 6.47). However, time of M genitalium testing affected the association (p value for effect-modification=0.004). For 29 case-control sets with endocervical samples tested 3 months prior to the first HIV-positive result, M genitalium infection increased the risk of HIV acquisition (crude OR=3.09; 95% CI 1.06 to 9.05, aOR=7.19; 95% CI 1.68 to 30.77), whereas there was little evidence of an association among the 13 case-control sets with samples tested at an earlier visit (crude OR=0.30: 95% CI 0.04 to 2.51; aOR=0.34; 95% CI 0.02 to 5.94). Conclusions Our study showed evidence of a temporal relationship between M genitalium infection and HIV acquisition that suggests that M genitalium infection may be a co-factor in the acquisition of HIV infection.

Macrolide resistance testing and molecular subtyping of Treponema pallidum strains from southern Africa

Objectives To determine whether the 23S ribosomal RNA (rRNA) A2058G and A2059G mutations that confer macrolide resistance are present among southern African strains of Treponema pallidum and to determine their subtype distribution. Methods 117 genital ulcer specimens, collected between March 2005 and April 2010 in South Africa and Lesotho and previously determined to be positive for T pallidum DNA by molecular testing, were retested using a commercial real-time PCR assay. Those specimens that were still positive for T pallidum DNA were screened for the macrolide resistance-encoding point mutations in the 23S rRNA gene using rapid PCR-based restriction digest assays. Molecular characterisation of two variable treponemal genes, arp and tpr, was used to subtype the T pallidum strains. Results 1 of 100 T pallidum-positive specimens, collected in Lesotho, contained the A2058G macrolide resistance-encoding 23S rRNA gene mutation, whereas the A2059G mutation was absent. It was possible to fully type 97/100 of all T pallidum DNA-positive samples. A total of nine arp repeat sizes, nine tpr patterns and a combined total of 20 subtypes were identified. Overall, the most common subtypes were 14d (32%), followed by 17d (12%), 14a (10%), 14b (8%), 22b (6%) and 14i (5%). Subtypes 14d and 14a were the predominant subtypes in samples from South Africa (43%) and Lesotho (22%), respectively. Conclusions Macrolide resistance among T pallidum strains appears to be uncommon in southern Africa. Although a high degree of genetic heterogeneity was observed among the strains tested, T pallidum subtype 14d appears to be the predominant circulating strain.

Natural history of Mycoplasma genitalium infection in a cohort of female sex workers in Kampala, Uganda

Background There have been few studies of the natural history of Mycoplasma genitalium in women. We investigated patterns of clearance and recurrence of untreated M. genitalium infection in a cohort of female sex workers in Uganda. Methods Women diagnosed as having M. genitalium infection at enrollment were retested for the infection at 3-month intervals. Clearance of infection was defined as testing negative after having a previous positive result: persistence was defined as testing positive after a preceding positive test result, and recurrence as testing positive after a preceding negative test result. Adjusted hazard ratios for M. genitalium clearance were estimated using Cox proportional hazards regression. Results Among 119 participants infected with M. genitalium at enrollment (prevalence, 14%), 55% had spontaneously cleared the infection within 3 months; 83%, within 6; and 93%, within 12 months. The overall clearance rate was 25.7/100 person-years (pyr; 95% confidence interval, 21.4–31.0). HIV-positive women cleared M. genitalium infection more slowly than did HIV-negative women (20.6/100 pyr vs. 31.3/100 pyr, P = 0.03). The clearance rate was slower among HIV-positive women with CD4 counts less than 350/mL3 than among those with higher CD4 counts (9.88/100 pyr vs. 29.5/100 pyr, P <; 0.001). After clearing the infection, M. genitalium infection recurred in 39% women. Conclusions M. genitalium is likely to persist and recur in the female genital tract. Because of the urogenital tract morbidity caused by the infection and the observed association with HIV acquisition, further research is needed to define screening modalities, especially in populations at high risk for HIV, and to optimize effective and affordable treatment options.

Prevalence and associations of genital ulcer and urethral pathogens in men presenting with genital ulcer syndrome to primary health care clinics in South Africa.

Background This study aimed to determine the prevalence of genital ulcer and urethral pathogens, as well as their association with clinical features, in men with genital ulcer disease (GUD) enrolled in a clinical trial. Methods Clinical data were collected by questionnaire. Ulcer swabs were tested for herpes simplex viruses (HSV-1/2), Treponema pallidum, Haemophilus ducreyi, and Chlamydia trachomatis L1-L3. First-pass urine was tested for urethral pathogens, namely Neisseria gonorrhoeae, C. trachomatis, Trichomonas vaginalis, and Mycoplasma genitalium. Pathogens were detected by real-time molecular assays. Blood was tested for HIV, HSV-2, and syphilis-associated antibodies. Pathogens and clinical associations were investigated using the &khgr;2 test. Results A total of 615 men with GUD were recruited. Herpes simplex virus (HSV-1, 4.2%; HSV-2, 98.2%) and bacterial pathogens were detected in 451 (73.6%) and 48 (7.8%) of genital ulcers, respectively. Human immunodeficiency virus, HSV-2, and treponemal antibodies were detected in 387 (62.9%), 434 (70.6%), and 141 (23.0%) men, respectively, whereas 54 men (8.8%) were rapid plasmin reagin (RPR) seropositive. A total of 223 urethral infections were diagnosed in 188 men (30.6%), including 69 (11.2%) M. genitalium, 64 (10.4%) T. vaginalis, 60 (9.8%) C. trachomatis, and 30 (4.9%) N. gonorrhoeae infections. Dysuria was reported by 170 men (27.6%), and 69 men (11.5%) had urethral discharge on examination. Urethral pathogens were detected in 102/409 (24.9%) men without these clinical features. Conclusions Herpes accounted for most GUD cases and urethral pathogen coinfections were common. Erythromycin, dispensed to treat infrequent chancroid and lymphogranuloma venereum cases, provided additional treatment of some asymptomatic urethral pathogens. Additional antibiotics would be required to treat asymptomatic trichomoniasis and gonorrhea.

Characterization of a novel β-lactamase-producing plasmid in Neisseria gonorrhoeae: sequence analysis and molecular typing of host gonococci

OBJECTIVES To determine the complete nucleotide sequence of the novel Johannesburg β-lactamase-encoding gonococcal plasmid (pEM1) and to determine the strain relatedness of Johannesburg plasmid-containing penicillinase-producing Neisseria gonorrhoeae (PPNG) by molecular typing. METHODS Eleven PPNG isolates containing the Johannesburg β-lactamase-encoding plasmid were previously identified among gonococci isolated from men with urethral discharge attending a clinic in Alexandra (Johannesburg) using a PCR assay. DNA sequence-based characterization of one such plasmid was performed to determine its relatedness to the prototype Asia plasmid. The 11 PPNG isolates containing the Johannesburg plasmid and 105 other clinical gonococci isolates were typed using N. gonorrhoeae multi-antigen sequence typing (NG-MAST). RESULTS Plasmid pEM1 was determined to comprise 4865 bp and to be a deletion derivative of the prototype Asia plasmid with a unique 2560 bp deletion in the non-TnA region. NG-MAST genotyping demonstrated a significant association between sequence type (ST) 502, or other closely related STs, and the Johannesburg plasmid-containing PPNG (P < 0.0001). CONCLUSIONS Sequencing of a novel β-lactamase-encoding plasmid (pEM1) found in PPNG isolates in Johannesburg shows it to be a deletion derivative of the prototype Asia plasmid, the deletion most likely arising as a result of DNA rearrangements. The majority of Johannesburg plasmid-containing PPNG isolates were, or were very closely related to, ST502.

Plasmid-mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates in South Africa: prevalence, detection and typing using a novel molecular assay.

Background: To detect and type plasmids responsible for penicillin and tetracycline resistance in Neisseria gonorrhoeae isolates using a novel duplex polymerase chain reaction (PCR) assay. Methods: A duplex PCR assay, to detect and type penicillinase-producing N. gonorrhoeae (PPNG), and plasmid-mediated tetracycline resistant N. gonorrhoeae (TRNG), was developed on the basis of published single assays. Gonococcal Isolate Surveillance Project control strains were used in assay development and then 209 consecutive N. gonorrhoeae isolates, collected from men with urethral discharge in 2008, were tested. Controls included Asia, Africa, and Toronto &bgr;-lactamase plasmids, as well as American and Dutch TRNG plasmids. PCR amplicons were detected using an Agilent 2100 Bioanalyzer. Minimum inhibitory concentrations (MIC) were determined with E tests. Penicillinase production was detected using Nitrocefin solution. Results: Among 209 gonococcal isolates, 54 (25.8%) PPNG and 154 (73.3%) TRNG were detected. The MIC50 and MIC90 values were determined for penicillin (0.19 and 32 mg/L) and tetracycline (6 and 16 mg/L). The assay detected the Africa-type (35.2%), the Toronto-type (44.4%), and a new type (20.3%) of &bgr;-lactamase plasmid. The American-type TRNG plasmid was 3-fold more frequent as compared with the Dutch-type. Although there was no overall association between the detection of PPNG and TRNG plasmids, only American type TRNG contained &bgr;-lactamase-encoding plasmids (P < 0.0001). Conclusions: The prevalence of plasmid-mediated resistance to tetracycline, and to a lesser extent penicillin, is high and neither drug is likely to have any future role in the treatment of gonorrhoea in South Africa. A novel &bgr;-lactamase plasmid was detected during the study and requires further characterization.

Mycoplasma genitalium Is Associated With Increased Genital HIV Type 1 RNA in Zimbabwean Women

BACKGROUND Mycoplasma genitalium is a common sexually transmitted infection associated with human immunodeficiency virus (HIV) infection. Some studies suggest that M. genitalium may increase the risk of HIV acquisition. However, results have been inconsistent, and this association has never been examined longitudinally. METHODS Stored endocervical samples from a longitudinal cohort study of 131 Zimbabwean women in whom HIV-1 seroconversion recently occurred were tested for detection and quantity of M. genitalium using polymerase chain reaction analysis. The associations between M. genitalium and the detection and quantity of genital HIV type 1 (HIV-1) RNA, the detection and quantity of plasma HIV-1 RNA, and the CD4(+) T-cell count was analyzed using mixed-effects regression analysis. RESULTS M. genitalium was detected in 10.5% of stored specimens (44 of 420), and infection persisted for up to 300 days. M. genitalium was independently associated with detection of genital HIV-1 RNA (adjusted odds ratio, 2.67; 95% confidence interval, .99-7.20), after adjustment for plasma viral load, viral set point, CD4(+) T-cell count, herpes simplex virus type 2 detection, and gonorrhea. There was no evidence of an association between M. genitalium detection or quantity and either plasma HIV-1 RNA load or CD4(+) T-cell count. CONCLUSIONS The growing evidence for an association between M. genitalium and HIV genital shedding and the high prevalence and persistence of M. genitalium in this population suggest that further research into this association is important. Consideration of the cost-effectiveness of M. genitalium screening interventions may be warranted.